The gastrointestine (i.e., G.I.) is an organ of the body that functions to physically, chemically and enzymatically process and break down ingested nutrients. The G.I. tract is also responsible for the uptake of nutrients into the body and the elimination of waste products. The G.I. tract consists of the stomach, which digests nutrients, stimulates other regions of the G.I. tract to secrete digestive enzymes, stores food temporarily, and releases chyme into the intestine at a controlled rate. The stomach also serves to secrete numerous chemicals and biological factors. However, the uptake of nutrients is not a significant function. Distal to the stomach is the duodenum, where neutralization of acidic chyme occurs. Surfactants, for the digestion of lipids, and proteases, for the degradation of proteins, are also secreted into the duodenum. As with the stomach, there is little absorption of nutrients in the duodenum. Uptake of nutrients, or more specifically their digestive products, takes place principally in the small intestine, comprising the jejunum and the ileum. On the other hand, the large intestine, consisting of the cecum the colon, are responsible for the storage of waste and water, and also for salt balance. There is little enzymatic activity in the large intestine, which is the least permeable section of the G.I. tract.
The majority of the surface area in the small and large intestines is made up of a layer of epithelial cells called enterocytes, which are specialized villus absorptive cells. The intestine is also lined with a mucus layer; Clamp, J. R., in Food Allergy and Intolerance, edited by J. Brostoff and S. J. Challacombe, pages 190-205 (1987). The mucus layer acts as a barrier to macromolecules, e.g., molecules having a molecular weight of greater than 17 kilodaltons; Thomson, A. B. R. and Dietschy, J. M. in Pharmacology of the Intestinal Permeation II, edited by T. Z. Czaky, page 20 (1984). The enterocyte layer, on the other hand, forms a tight lipid barrier to smaller molecules, i.e., peptides of about 500 daltons or so; Smith, P. L. et al., Volume 8, pages 253-290 (1992). Thus, the lining of the intestine serves as an efficient barrier to both lipophilic and hydrophilic molecules. As a consequence, the oral administration of a large, macromolecular therapeutic such as a protein is normally limited as to effectiveness.
However, some molecules are specifically taken up in the G.I. tract as a normal function of the digestive process. These substances include amino acids, glucose and vitamins, among others. For such molecules, native biological mechanisms for transportation across the intestinal lining exist. In particular, amino acids and glucose are taken up by transporter molecules located in the lumenal or apical membrane domain of enterocytes. Receptors for vitamin uptake are also present in the apical domain of the enterocyte lining.
Of special interest here is the biological mechanism for the uptake of vitamin B.sub.12 ("VB.sub.12 "). VB.sub.12, also known as cyanocobalamin, is composed of a corrin ring structure which surrounds an atom of cobalt. VB.sub.12 is normally ingested through animal products and released into the acidic environment of the stomach. A transport protein for VB.sub.12, called intrinsic factor ("IF"), is also secreted into the lumen of the stomach in humans by parietal cells; Levine, J. S. et al., Gastroenterology, Volume 79, pages 493-502 (1980). IF, a glycoprotein of about 44 kilodaltons, is typically released in amounts far in excess of those needed to promote the physiological absorption of VB.sub.12. Once secreted, IF binds to VB.sub.12 with high affinity (K.sub.a 1.9.times.10.sup.12 M.sup.-1) , but only under the neutral conditions of pH present in the duodenum. After IF becomes complexed with VB.sub.12 it becomes resistant to the proteases present in that organ which degrade most proteins; Allen, R. H. et al., Journal of Clinical Investigation, Volume 61, pages 47-54 (1978).
Receptors that bind to the IF-VB.sub.12 complex are present in the apical membrane domain of enterocytes, predominantly in the ileum; Hagedorn, C. H. and Alpers, D. H., Gastroenterology, Volume 73, pages 1019-1022(1977). While the number of receptors for the IF-VB.sub.12 complex on each enterocyte is small, i.e., about 300-400 per cell, the binding affinity for the IF-VB.sub.12 complex is high, 4.0.times.10.sup.9 M.sup.-1 ; Mathan, V. I. et al., Journal of Clinical Investigation, Volume 54, pages 598-608 (1974). After binding to its receptor, the IF-VB.sub.12 complex is internalized in the enterocyte cell body; Kapadia, C. R. and Donaldson, R. M., Gastroenterology, Volume 76, page 1163P (1979). IF is apparently then degraded in the enterocyte. VB.sub.12, on the other hand, is trancytosed across the cell and then, in a complex with the serum transporting protein, transcobalamin II (TCII), is released into the systemic circulation; Rothenberg, S. P. et al., British Journal of Haemotology, Volume 40, page 401 (1978), and Dix, C. J. et al., Gastroenterology, Volume 98, pages 1272-1279 (1990).
It has been proposed that this VB.sub.12 uptake mechanism may be utilized to transport biologically active substances such as drugs, hormones, antigenic material, and the like, from the intestinal lumen into circulatory blood by covalently coupling these substances to VB.sub.12. Published European patent application 0 220 030 A2 discloses a process for the preparation of VB.sub.12 -polypeptide conjugates involving the acid hydrolysis of amide groups on the propionamide side chains adjacent to rings A, B and C of VB.sub.12, followed by chemical linking to amino groups of the polypeptide through the use of a carbodiimide. The synthesis of conjugates of VB.sub.12 with bovine serum albumin (BSA), neomycin sulfate and a D-lys-6 analog of lutenizing hormone releasing hormone (LHRH) is exemplified in the application. In addition, the oral administration of VB.sub.12 -BSA and VB.sub.12 -lys-6-LHRH conjugates to mice is demonstrated. See, also, Marques et al., Inorganica Chimica Acta, Volume 162, pages 151-155 (1989).